Gastrin receptor-avid peptide conjugates

ABSTRACT

A compound for use as a therapeutic or diagnostic radiopharmaceutical includes a group capable of complexing a medically useful metal attached to a moiety which is capable of binding to a gastrin releasing peptide receptor. A method for treating a subject having a neoplastic disease includes administering to the subject an effective amount of a radiopharmaceutical having a metal chelated with a chelating group attached to a moiety capable of binding to a gastrin releasing peptide receptor expressed on tumor cells with subsequent internalization inside of the cell. A method of forming a therapeutic or diagnostic compound includes reacting a metal synthon with a chelating group covalently linked with a moiety capable of binding a gastrin releasing peptide receptor.

GRANT REFERENCE

[0001] The research carried out in connection with this invention wassupported in part by a grant from the Department of Energy (DOE), grantnumber DE-FG02-89ER60875, a grant from the U.S. Department of VeteransAffairs Medical Research Division and the Department of RadiologyMU-C2-02691. The Government has certain rights in the invention.

[0002] This application is based on Provisional Application which wasfiled on Apr. 22, 1997, Serial No. 60/044,049.

TECHNICAL FIELD

[0003] This invention relates to radionuclide-labeled compounds usefulas radiopharmaceuticals. More particularly, the present inventionrelates to conjugates of bombesin (BBN) analogues and a metal complexinggroup which, when complexed to a radionuclide, are useful therapeuticand imaging agents for cancer cells that express gastrin releasingpeptide (GRP) receptors.

BACKGROUND OF THE INVENTION

[0004] Detection and treatment of cancers using radiopharmaceuticalsthat selectively target cancers in human patients has been employed forseveral decades. Unfortunately, only a limited number of site-directedradiopharmaceuticals that exhibit highly specific in vivo localizationin or near cancer cells are currently in routine use, as being approvedby the United States Food and Drug Administration (FDA). There is agreat deal of interest in developing new radioactive drugs due to theemergence of more sophisticated biomolecular carriers that have highaffinity and high specificity for in vivo targeting of tumors. Severaltypes of agents are being developed and have been investigated includingmonoclonal antibodies (MAbs), antibody fragments (F_(AB)'s and(F_(AB))₂'s), receptor-avid peptides [Bushbaum, 1995; Fischman et al.,1993; Schubiger et al. 1996].

[0005] The potential utility of using radiolabeled receptor-avidpeptides for producing radiopharmaceuticals is best exemplified by¹¹¹In-DTPA-conjugated octreotide (an FDA approved diagnostic imagingagent, Octreoscan®, marketed in the United States. by MallinckrodtMedical, Inc.) [Lowbertz et al. 1994]. This radiopharmaceutical is an¹¹¹In-DTPA conjugate of Octreotide, a small peptide analogue of thehuman hormone somatostatin. This drug specifically binds to somatostatinreceptors that are over-expressed on neuroendocrine cancers (e.g.,carcinoid Ca, neuroblastoma, etc.) as well as others [Krenning et al.,1994]. Since indium-111 (¹¹¹In) is not the ideal radionuclide forscintigraphic imaging, other somatostatin analogues and otherreceptor-avid biomolecules that are labeled with ^(99m)Tc (the optimalradionuclide for diagnostic imaging) are being studied and developed[Eckelman, 1995; Vallabhajosula et al., 1996].

[0006] Bombesin (BBN) is a 14 amino acid peptide that is an analogue ofhuman gastrin releasing peptide (GRP) that binds to GRP receptors withhigh specificity and has an affinity similar to GRP [Davis et al.,1992]. GRP receptors have been shown to be over-expressed or uniquelyexpressed on several types of cancer cells. Binding of GRP receptoragonists (also autocrine factors) increases the rate of cell division ofthese cancer cells. For this reason, a great deal of work has been, andis being pursued to develop BBN or GRP analogues that are antagonists[Davis et al., 1992; Hoffken, 1994; Moody et al., 1996; Coy et al.,1988; Cai et al., 1994]. These antagonists are designed to competitivelyinhibit endogenous GRP binding to GRP receptors and reduce the rate ofcancer cell proliferation [Hoffken, 1994]. Treatment of cancers usingthese antagonists with these non-radioactive peptides requires chronicinjection regimens (e.g., daily, using large quantities of the drug).

[0007] In designing an effective receptor-avid radiopharmaceutical foruse as a diagnostic or therapeutic agent for cancer, it is importantthat the drug have appropriate in vivo targeting and pharmacokineticproperties [Fritzberg et al., 1992; Eckelman et al., 1993]. For example,it is essential that the radiolabeled receptor-avid peptide have highspecific uptake by the cancer cells (e.g., via GRP receptors). Inaddition, it is necessary that once the radionuclide localizes at atumor site, it must remain there for an extended time to deliver ahighly localized radiation dose to the tumor. In order to achievesufficiently high specific uptake of radiolabeled BBN analogues intumors, the binding affinity of promising derivatives must be high(i.e., K_(d)≅1-5 nmolar or less) with prolonged retention ofradioactivity [Eckelman et al., 1995; Eckelman, et al., 1993]. Work with¹²⁵I-BBN derivatives has shown, however, that for cancer cells that bindthe ¹²⁵I-BBN derivatives (whether they be agonists or antagonists), theradioactivity is either washed off or expelled from the cells (in vitro)at a rapid rate [Hoffman et al., 1997]. Thus, these types of derivativeshave a low probability of remaining “trapped” at the tumor site (invivo) sufficiently long to be effective therapeutic or diagnosticagents.

[0008] Developing radiolabeled peptides that are cleared efficientlyfrom normal tissues is also an important and especially critical factorfor therapeutic agents. When labeling biomolecules (e.g., MAb, F_(AB)'sor peptides) with metallic radionuclides (via a chelate conjugation), alarge percentage of the metallic radionuclide (in some chemical form)usually becomes “trapped” in either the kidney or liver parenchyma(i.e., is not excreted into the urine or bile) [Duncan et al., 1997;Mattes, 1995]. For the smaller radiolabeled biomolecules (i.e., peptidesor FAB's), the major route of clearance of activity is through thekidneys which in turn retain high levels of the radioactive metal (i.e.,normally >10-15% of the injected dose) [Duncan et al., 1997]. Thispresents a major problem that must be overcome in the development oftherapeutic agents that incorporate metallic radionuclides, otherwisethe radiation dose to the kidneys would be excessive. For example,¹¹¹In-octreotide, the FDA approved diagnostic agent, exhibits highuptake and retention in kidneys of patients [Eckelman et al., 1995].Even though the radiation dose to the kidneys is higher than desirable,it is tolerable in that it is a diagnostic radiopharmaceutical (it doesnot emit alpha- or beta-particles), and the renal dose does not produceobservable radiation induced damage to the organ.

[0009] It has now been found that conjugating BBN derivatives which areagonists in non-metallated conjugates whichthat exhibit bindingaffinities to GRP receptors that are either similar to or approximatelyan order of magnitude lower than the parent BBN derivative. [Li et al.,1996a] These data coupled with our recent results show that it is nowpossible to add radiometal chelates to BBN analogues, which areagonists, and retain GRP receptor binding affinities that aresufficiently high (i.e., approx. 1-5 nmolar K_(d)'s) for furtherdevelopment as potential radiopharmaceuticals. These agonist conjugatesare transported intracellularly after binding to cell surface GRPreceptors and retained inside of the cells for extended time periods. Inaddition, in vivo studies in normal mice have shown that retention ofthe radioactive metal in the kidneys was low (i.e., <5%) with themajority of the radioactivity excreted into the urine.

[0010] According to one aspect of the present invention, there isprovided a BBN conjugate consisting of essentially a radio-metal chelatecovalently appended to the receptor binding region of BBN [e.g.,BBN(8-14)] to form radiolabeled BBN analogues that have high specificbinding affinities with GRP receptors. These analogues are retained forlong times inside of GRP expressing cancer cells. Furthermore, theirclearance from the bloodstream, into the urine with minimal kidneyretention, is efficient. Preferably, the radiometals are selected from^(99m)Tc, ^(186/188)Re, ¹⁰⁵Rh, ¹⁵³Sm, ¹⁶⁶Ho, ⁹⁰Y or ¹⁹⁹Au, all of whichhold the potential for diagnostic (i.e., ^(99m)Tc) or therapeutic (i.e.,^(186/188)Re, ¹⁰⁵Rh, ¹⁵³Sm, ¹⁶⁶Ho, ⁹⁰Y, and ¹⁹⁹Au) utility in cancerpatients [Schubiger et al, 1996; Eckelman, 1995; Troutner, 1978].

SUMMARY OF THE INVENTION

[0011] In accordance with the present invention, there is provided acompound for use as a therapeutic or diagnostic radiopharmaceuticalwhich includes a group which is capable of complexing a metal attachedto a moiety capable of binding to a gastrin releasing peptide receptor.

[0012] Additionally, in accordance with the present invention, a methodfor treating a subject having a neoplastic disease which includes thestep of administering to the subject an effective amount of aradiopharmaceutical having a metal chelated with a chelating groupattached to a moiety capable of binding to a gastrin releasing peptidereceptor on a cancer cell, subsequently intracellularly transported andresidualized inside the cell, is disclosed.

[0013] Additionally, in accordance with the present invention, a methodof forming, a therapeutic or diagnostic compound including the step ofreacting a metal synthon with a chelating group covalently linked with amoiety capable of binding a gastrin releasing peptide receptor isdisclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Other advantages of the present invention will be readilyappreciated as the same becomes better understood by reference to thefollowing detailed description when considered in connection with theaccompanying drawings wherein:

[0015]FIG. 1 illustrates a radiometal conjugate according to the presentinvention;

[0016]FIG. 2 is an ORTEP drawing of the {Rh[16]aneS₄-olCl₂}⁺,illustrating the crystal structure a Rhodium macrocycle;

[0017]FIG. 3 illustrates a coupling reaction wherein a spacer group iscoupled to a bombesin agonist binding moiety;

[0018]FIG. 4 illustrates a coupling reaction for coupling a metalchelate to a peptide;

[0019]FIG. 5 illustrates several iodinated bombesin analogues includingtheir IC₅₀'s;

[0020]FIG. 6 illustrates several tethered bombesin analogues;

[0021]FIG. 7 illustrates several [16]aneS₄ bombesin analogues;

[0022]FIG. 8 is a graph illustrating IC₅₀ analysis wherein %-I-125-BBNtotal uptake versus molar concentration of displacing ligand is shown;

[0023]FIG. 9 illustrates several Rhodium-[16]aneS₄ bombesin analogues;

[0024]FIG. 10 illustrates an HPLC chromatogram of Rhodium-BBN-37 wherein(A) illustrates ¹⁰⁵RhCl₂-BBN-37 and (B) illustrates RhCl₂-BBN-37;

[0025]FIG. 11 is a graph illustrating ¹²⁵I-Tyr⁴-bombesin internalizationefflux from Swiss 3T3 cells;

[0026]FIG. 12 illustrates I-125 bombesin internalization efflux in I-125free buffer wherein ¹²⁵I-Tyr⁴-BBN vs. ¹²⁵I-Lys³-BBN efflux from Swiss3T3 cells is shown;

[0027]FIG. 13 is a graph illustrating the efflux of ¹⁰⁵Rh-BBN-37 fromSwiss 3T3 cells;

[0028]FIG. 14 illustrates several ¹⁰⁵Rhodium bombesin analoguesincluding their IC₅₀'s;

[0029]FIG. 15 is a graph illustrating ¹⁰⁵Rh-BBN-61 efflux from Swiss 3T3cells;

[0030]FIG. 16 is a graph illustrating the efflux of ¹⁰⁵Rh-BBN-22 vs.¹⁰⁵Rh-BBN-37 from Swiss 3T3 cells;

[0031]FIG. 17 are graphs illustrating Pancreatic CA cell binding wherein(A) illustrates the efflux ¹²⁵I-Tyr⁴-BBN from CF PAC1 cells and (B)illustrates the efflux of ¹⁰⁵Rh-BBN-37 from CF PAC1 cells; and

[0032]FIG. 18 are graphs illustrating Prostate CA cell binding wherein(A) illustrates the efflux of ¹²⁵I-Tyr⁴-BBN from PC-3 cells and (B)illustrates the efflux of ¹⁰⁵Rh-BBN-37 from PC-3 cells.

[0033]FIG. 19 illustrates 5 [16]aneS₄ bombesin analogues.

[0034]FIG. 20 illustrates 4 Rhodium-[16]aneS₄ bombesin analogues.

[0035]FIG. 21 illustrates 3 different N₃S-BFCA conjugates of BBN(7-14).

[0036]FIG. 22 illustrates on HPLC chromatogram of ^(99m)Tc-BBN-122.

[0037]FIG. 23 is a graph illustrating ^(99m)TC-BBN-122 internalizationefflux from human prostate cancer cells (PC-3 cells).

[0038]FIG. 24 is a graph illustrating ^(99m)Tc-BBN-122 internalizationefflux from human pancreatic tumor cells (CFPAC-1 cells).

[0039]FIG. 25 is a graph illustrating ^(99m)Tc-RP-414-BBN-42 retentionin PC-3 prostate cancer cells.

[0040]FIG. 26 is a graph illustrating 99m Tc-42 retention in CFPAC-1pancreatic cancer cells.

DETAILED DESCRIPTION OF THE INVENTION

[0041] According to the present invention, compounds for use asdiagnostic and/or therapeutic radiopharmaceuticals include a groupcapable of completing a metal attached to a moiety capable of binding toa gastrin releasing peptide (GRP) receptor as shown in FIG. 1. Themoiety capable of specific binding to the GRP receptor is a GRP agonist.A GRP agonist would activate or produce response by the GRP receptorupon interaction with the GRP receptor and would be subsequentlyinternalized inside of the cell by endocytosis. In contrast, a GRPantagonist would counteract the effect of an agonist and would not beinternalized inside of the cell.

[0042] More specifically, the GRP agonist is a compound such as selectedamino acid sequences or peptidomimetics which are known to activate thecell following binding with high affinity and selectivity to GRPreceptors and that can be covalently linked to the metal complexinggroup. Many examples of specific modifications of the BBN(8-14) that canbe made to produce sequences with high antagonistic and agonisticbinding affinity for GRP repectors have been reported by numerousinvestigations [Davis et al., 1992; Hoffken, 1994; Moody et al., 1996;Coy et al., 1988; Cai et al., 1994; Moody et al., 1995; Leban et al.,1994; Cai et al., 1992].

[0043] In a preferred embodiment of the present invention, the metalcomplexing group or moiety is a chelating agent or chelator whichcomplexes to metals such as ¹⁰⁵Rh—, ^(186/188)Re—, ^(99m)Tc, ¹⁵³Sm,¹⁶⁶Ho, ⁹⁰Y or ¹⁹⁹Au. The chelating agent or chelator is attached orbound to the GRP agonist “binding region” to produce a conjugate thatretains its capability for high affinity and specific binding to GRPreceptors.

[0044] In a more preferred embodiment of the present invention, the GRPagonist is a bombesin (BBN) analogue and/or a derivative thereof. TheBBN derivative or analog thereof preferably contains either the sameprimary structure of the BBN binding region [i.e., BBN(8-14)] or similarprimary structures, with specific amino acid substitutions, that willspecifically bind to GRP receptors with better or similar bindingaffinities as BBN alone (i.e., K_(d)≅1-5 nmolar) Compounds containingthis BBN binding region (or binding moiety), when covalently linked toother groups (e.g., a radiometal chelate), are also referred to as BBNconjugates.

[0045] In general, the compounds of the present invention have astructure of the general formula:

X—Y—B

[0046] wherein X is a group capable of complexing a metal, such as aradiometal; Y is a covalent bond on a spacer group; and B is a bombesinagonist binding moiety.

[0047] The metal bound to the metal complexing group can be any suitablemetal chosen for a specific therapeutic or diagnostic use includingtransition metals and γ and β emitting isotopes. Preferably, the metalis a radiometal such as ¹⁰⁵Rh—, ^(99m)Tc—, ^(186/188)Re, ¹⁵³Sm—, ¹⁶⁶Ho—,⁹⁰Y—, and ¹⁹⁹Au— whose chelates can be covalently linked (i.e.,conjugated) to the specific BBN binding region via the N-terminal end ofthe primary binding sequence (e.g., BBN-8 or Trp⁸) as shown in FIG. 1.

[0048] In a preferred embodiment, the radiometal complexes arepositioned by being spaced apart from or remotely from the amino acidTrp⁸ by the spacer groups. The spacer groups can include a peptide(i.e., ≧1 amino acid in length), a hydrocarbon spacer of C₁-C₁₀ or acombination of thereof. Preferably, the hydrocarbon spacer has is aC₃-C₉ group. The resulting radio-labeled BBN conjugates retain highbinding affinity and specificity for GRP receptors and are subsequentlyinternalized inside of the cell.

[0049] The BBN conjugates can further incorporate a spacer group orcomponent to couple the binding moiety to the metal chelator (or metalbinding backbone) while not adversely affecting either the targetingfunction of the BBN-binding moiety or the metal complexing function ofthe metal chelating agent.

[0050] The term “spacer group” or “linker” refers to a chemical groupthat serves to couple the BBN binding moiety to the metal chelator whilenot adversely affecting either the targeting function of the BBN bindingmoiety or the metal complexing function of the metal chelator. Suitablespacer groups include peptides (i.e., amino acids linked together)alone, a non-peptide group (e.g., hydrocarbon chain) or a combination ofan amino acid sequence and a non-peptide spacer. The type of spacergroup used in most of the experimental studies described below in theExamples section were composed of a combination of L-glutamine andhydrocarbon spacers. A pure peptide spacer could consist of a series ofamino acids (e.g., diglycine, triglycine, gly-gly-glu, etc.), in whichthe total number of atoms between the N-terminal residue of the BBNbinding moiety and the metal chelator in the polymeric chain is ≦12atoms.

[0051] The spacer can also include a hydrocarbon chain [i.e.,R₁—(CH₂)_(n)—R₂] wherein n is 0-10, preferably n=3 to 9, R₁ is a group(e.g., H₂N—, HS—, —COOH) that can be used as a site for covalentlylinking the ligand backbone or the preformed metal chelator or metalcomplexing backbone; and R₂ is a group that is used for covalentcoupling to the N-terminal NH₂-group of the BBN binding moiety (e.g., R₂is an activated COOH group). Several chemical methods for conjugatingligands (i.e., chelators) or preferred metal chelates to biomoleculeshave been well described in the literature [Wilbur, 1992; Parker, 1990;Hermanson, 1996; Frizberg et al., 1995]. One or more of these methodscould be used to link either the uncomplexed ligand (chelator) or theradiometal chelate to the spacer group or to link the spacer group tothe BBN(8-14) derivatives. These methods include the formation of acidanhydrides, aldehydes, arylisothiocyanates, activated esters, orN-hydroxysuccinimides [Wilbur, 1992; Parker, 1990; Hermanson, 1996;Frizberg et al., 1995].

[0052] The term “metal complexing chelator” refers to a molecule thatforms a complex with a metal atom that is stable under physiologicalconditions. That is, the metal will remain complexed to the chelatorbackbone in vivo. More particularly, a metal complexing chelator is amolecule that complexes to a radionuclide metal to form a metal complexthat is stable under physiological conditions and which also has atleast one reactive functional group for conjugation with the BBN agonistbinding moiety. Metal complexing chelators can include monodentate andpolydentate chelators [Parker, 1990; Frizberg et al., 1995; Lister-Jameset al., 1997; Li et al., 1996b; Albert et al., 1991; Pollak et al.,1996; de Jong et al., 1997; Smith et al., 1997]. Metal complexingchelators include tetradentate metal chelators which can be macrocyclicand have a combination of four nitrogen and/or sulphurmetal-coordinating atoms [Parker et al., 1990; Li et al., 1996b] and aredesignated as N₄, S₄, N₃S, N₂S₂, NS₃, etc. as shown in FIG. 2. A numberof suitable multidentate chelators that have been used to conjugateproteins and receptor-avid molecules have been reported [Frizberg etal., 1995; Lister-James et al., 1997; Li et al., 1996b; Albert et al.,1991; Pollak et al., 1996; de Jong et al., 1997]. These multidentatechelators can also incorporate other metal-coordinating atoms such asoxygen and phosphorous in various combinations. The metal bindingcomplexing moiety can also include “3+1” chelators [Seifert et al.,1998].

[0053] For diagnostic purposes, metal complexing chelators preferablyinclude chelator backbones for complexing the radionuclide metal^(99m)Tc. For therapeutic purposes, metal complexing chelatorspreferably include chelator backbones that complex the radionuclideMetals ¹⁰⁵Rh, ^(186/188)Re, ¹⁵³Sm, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁹⁹Au [Schubiger etal., 1996; Hoffken, 1994].

[0054] As was briefly described above, the term “bombesin agonist” or“BBN agonist” refers to compounds that bind with high specificity andaffinity to GRP receptors, and upon binding to the GRP receptor, areintracellularly internalized. Suitable compounds include peptides,peptidomimetics and analogues and derivatives thereof. In particular,previous work has demonstrated that the region on the BBN peptidestructure required for binding to GRP receptors spans from residue 8through 14 [Davis et al., 1992; Hoffken, 1994; Moody et al., 1996; Coy,1988; Cai et al., 1994]. The presence of methionine (Met) at positionBBN-14 will generally confer agonistic properties while the absence ofthis residue at BBN-14 generally confers antagonistic properties[Hoffken, 1994].

[0055] It is well documented in the art that there are a few andselective number of specific amino acid substitutions in the BBN (8-14)binding region (e.g., D-Ala¹¹ for L-Gly¹¹ or D-Trp⁸ for L-Trp⁸), whichcan be made without decreasing binding affinity [Leban et al., 1994; Qinet al., 1994; Jensen et al., 1993]. In addition, attachment of someamino acid chains or other groups to the N-terminal amine group atposition BBN-8 (i.e., the Trp⁸ residue) can dramatically decrease thebinding affinity of BBN analogues to GRP receptors [Davis et al., 1992;Hoffken, 1994; Moody et al., 1996; Coy, et al., 1988; Cai et al., 1994].In a few cases, it is possible to append additional amino acids orchemical moieties without decreasing binding affinity. The effects ofconjugating various side chains to BBN-8 on binding affinity, therefore,is not predicable.

[0056] The BBN conjugates of the present invention can be prepared byvarious methods depending upon the selected chelator. The peptideportion of the conjugate can be most conveniently prepared by techniquesgenerally established and known in the art of peptide synthesis, such asthe solid-phase peptide synthesis (SPPS) approach. Solid-phase peptidesynthesis (SPPS) involves the stepwise addition of amino acid residuesto a growing peptide chain that is linked to an insoluble support ormatrix, such as polystyrene. The C-terminal residue of the peptide isfirst anchored to a commercially available support with its amino groupprotected with an N-protecting agent such as a t-butyloxycarbonyl group(tBoc) or a fluorenylmethoxycarbonyl (FMOC) group. The amino protectinggroup is removed with suitable deprotecting agents such as TFA in thecase of tBOC or piperidine for FMOC and the next amino acid residue (inN-protected form) is added with a coupling agent such asdicyclocarbodiimide (DCC). Upon formation of a peptide bond, thereagents are washed from the support. After addition of the finalresidue, the peptide is cleaved from the support with a suitable reagentsuch as trifluoroacetic acid (TFA) or hydrogen fluoride (HF).

[0057] The spacer groups and chelator components are then coupled toform a conjugate by reacting the free amino group of the Trp⁸ residue ofthe BBN binding moiety with an appropriate functional group of thechelator, metal chelator or spacer group, such as a carboxyl group oractivated ester.

[0058] The BBN conjugate can also incorporate a metal complexingchelator backbone that is peptidic and compatible with solid-phasepeptide synthesis. In this case, the chelator backbone can be added tothe BBN binding moiety in the same manner as described above or, moreconveniently, the metal complexing chelator backbone coupled to the BBNbinding moeity can be synthesized in toto starting from the C-terminalresidue of the peptide and ending with the N-terminal residue of themetal complexing chelator structure.

[0059] The chelator backbones used in accordance with the presentinvention are commercially available or they could be made by methodssimilar to those outlined in the literature [Frizberg et al., 1995;Lister-James et al., 1997; Li et al., 1996b; Albert et al., 1991; Pollaket al., 1996; de Jong et al., 1997; Smith et al., 1997; Seifert et al.,1998]. Attachment of the spacer groups to functionalizable atomsappended to the ligand backbone can be performed by standard methodsknown to those skilled in the art. For example, the HOBt/HBTU activated-COOH group on 5-aminovaleric acid (5-AVA) was reacted with theN-terminal amine on Gln⁷ to produce an amide linkage as shown in FIG. 3.Similarly, the —COOH group attached to the characterized [16]aneS₄ligand was conjugated to the amine group on the hydrocarbon spacer(shown below) by reaction of the HOBt/HBTU activated carboxyl groupappended to the [16]aneS₄ macrocycle with the terminal amine group on5-AVA to form BBN-37 as shown in FIG. 4. Other standard conjugationreactors that produce covalent linkages with amine groups can also beused [Wilbur, 1992; Parker, 1990].

[0060] The chelating framework, conjugated via Trps, complexes theradiometals should form a 1:1 chelator to metal ratio. Since ^(99m)Tchas a short half-life (6 hour) and is a diagnostic radionuclide, themethod of forming the ^(99m)Tc-BBN analogues should permit complexation(either directly or by transmetallation) of ^(99m)Tc to the conjugatedchelating framework in a one-step, high yield reaction (exemplifiedbelow in the Experimental Section).

[0061] In contrast, the longer half lives of the therapeuticradionuclides (e.g., ¹⁰⁵Rh, ^(186/188)Re, ¹⁵³Sm, ¹⁶⁶Ho, ⁹⁰Y, or ⁹⁹Au)permit formation of the corresponding radiolabeled BBN analogues byeither a one step high yield complexation step or by preforming a ¹⁰⁵Rh,^(186/188)Re, ¹⁵³Sm, ¹⁶⁶Ho, ⁹⁰Y or ¹⁹⁹Au chelate synthon followed byconjugation of the preformed complex to the N-terminal end of the BBNbinding moiety. In all cases, the resulting specific activity of thefinal radiolabeled BBN derivative must be high (i.e., >1 Ci/μmole).

[0062] Re- and Tc-Conjugates

[0063] Re and Tc are both in row VIIB of the Periodic Table and they arechemical congeners. Thus, for the most part, the complexation chemistryof these two metals with ligand frameworks that exhibit high in vitroand in vivo stabilities are the same [Eckelman, 1995]. Many ^(99m)Tc or^(186/188)Re complexes, which are employed to form stable radiometalcomplexes with peptides and proteins, chelate these metals in their +5oxidation state [Lister-James et al., 1997]. This oxidation state makesit possible to selectively place ^(99m)Tc- or ^(186/188)Re into ligandframeworks already conjugated to the biomolecule, constructed from avariety of ^(99m)Tc(V) and/or ^(186/188)Re(V) weak chelates (e.g.,^(99m)Tc-glucoheptonate, citrate, gluconate, etc.) [Eckelman, 1995;Lister-James et al., 1997; Pollak et al., 1996]. Tetradentate ligandframeworks have been shown to form well-defined, single chemical speciesin high specific activities when at least one thiol group or at leastone hydroxymethylene phosphine group is present on the ligand backbone[Smith et al., 1997].

[0064] Ligands which form stable Tc(V) or Re(V) tetradentate complexescontaining, but not limited to, amino N-atoms, amido-N-atoms,carboxy-O-atoms and thioether-S-atoms, are donor atoms that can also bepresent [Eckelman, 1995; Fritzberg et al., 1992; Parker, 1990; Frizberget al., 1995; Pollak et al., 1996; Seifert et al., 1998]. Depending uponthe mix of donor atoms (groups), the overall complex charge normallyranges from −1 to +1.

[0065] Incorporation of the metal within the conjugate can be achievedby various methods commonly known in the art of coordination chemistry.When the metal is technetium-99m, the following general procedure can beused to form a technetium complex. A peptide-chelator conjugate solutionis formed by initially dissolving the conjugate in aqueous alcohol suchas ethanol. The solution is then degassed to remove oxygen. When an —SHgroup is present in the peptide, the thiol protecting group are removedwith a suitable reagent, for example with sodium hydroxide, and are thenneutralized with an organic acid such as acetic acid (pH 6.0-6.5). Inthe labeling step, sodium pertechnetate obtained from a molybdenumgenerator is added to a solution of the conjugate with a sufficientamount of a reducing agent, such as stannous chloride, to reducetechnetium and is then heated. The labeled conjugate can be separatedfrom the contaminants ^(99m)TcO₄ ⁻ and colloidal ^(99m)TcO₂chromatographically, for example with a C-18 Sep Pak cartridge[Millipore Corporation, Waters Chromatography Division, 34 Maple Street,Milford, Mass. 01757].

[0066] In an alternative method, the labeling can be accomplished by atranschelation reaction. The technetium source is a solution oftechnetium complexed with labile ligands facilitating ligand exchangewith the selected chelator. Examples of suitable ligands fortranschelation includes tartrate, citrate, gluconate, andheptagluconate. It will be appreciated that the conjugate can be labeledusing the techniques described above, or alternatively, the chelatoritself may be labeled and subsequently coupled to the peptide to formthe conjugate; a process referred to as the “prelabeled chelate” method.

[0067] When labeled with diagnostically and/or therapeutically usefulmetals, peptide-chelator conjugates or pharmaceutically acceptablesalts, esters, amides, and prodrugs of the present invention can be usedto treat and/or detect cancers, including tumors, by proceduresestablished in the art of radiodiagnostics and radiotherapeutics.[Bushbaum, 1995; Fischman et al., 1993; Schubiger et al., 1996; Lowbertzet al., 1994; Krenning et al., 1994]. A conjugate labeled with aradionuclide metal, such as technetium-99m, can be administered to amammal, including human patients or subjects, by intravenous orintraperitoneal injection in a pharmaceutically acceptable carrierand/or solution such as salt solutions like isotonic saline. The amountof labeled conjugate appropriate for administration is dependent uponthe distribution profile of the chosen conjugate in the sense that arapidly cleared conjugate may be administered in higher doses than onethat clears less rapidly. Unit doses acceptable for Tc-99m imagingradiopharmaceuticals inflammation are in the range of about 5-40 mCi fora 70 kg individual. In vivo distribution and localization can be trackedby standard scintigraphic techniques at an appropriate time subsequentto administration; typically between thirty minutes and 180 minutesdepending upon the rate of accumulation at the target site with respectto the rate of clearance at non-target tissue.

[0068] The compounds of the present invention can be administered to apatient alone or as part of a composition that contains other componentssuch as excipients, diluents, and carriers, all of which are well-knownin the art. The compounds can be administered to patients eitherintravenously or intraperitoneally.

[0069] There are numerous advantages associated with the presentinvention. The compounds made in accordance with the present inventionforms a stable, well-defined ^(99m)Tc or ^(186/188)Re conjugateanalogues of BBN agonists. Similar BBN against analogues can also bemade by using appropriate chelator frameworks for the respectiveradiometals, to form stable-well-defined products labeled with ¹⁵³Sm,⁹⁰Y, ¹⁶⁶Ho, ¹⁰⁵Rh or ¹⁹⁹Au. The radiolabeled BBN agonist conjugatesselectively bind to neoplastic cells expressing GRP receptors becomeinternalized and are retained in the tumor cells for extended timeperiods. Incorporating the spacer group between the metal chelator andthe BBN agonist binding moiety maximizes the uptake and retention of theradioactive metal inside of the neoplasts or cancer cells. Theradioactive material that does not reach (i.e., does not bind) thecancer cells is preferentially excreted efficiently into the urine withminimal radiometal retention in the kidneys.

[0070] The following examples are presented to illustrate specificembodiments and demonstrate the utility of the present invention.

[0071] Experimental Section

EXAMPLE I Synthesis and in vitro Binding Assessment of Synthetic BBNAnalogues Employing Hydrocarbon Chain Spacers

[0072] A. Synthesis:

[0073] Many BBN analogues were synthesized by Solid Phase PeptideSynthesis (SPPS). Each peptide was prepared by SPPS using an AppliedBiosystems Model 432A peptide synthesizer. After cleavage of each BBNanalogue from the resin using Trifluoracetic acid (TFA), the peptideswere purified by C₁₈ reversed-phase HPLC using a Vydac HS54 column andCH₃CN/H₂O containing 0.1% TFA as the mobile phase. After collection ofthe fraction containing the desired BBN peptide (approx. 80-90% yield inmost cases), the solvent was evaporated. The identity of each BBNpeptide was confirmed by FAB-mass spectrometry, Department ofChemistry—Washington University, St. Louis, Mo.

[0074] Various amino acid sequences (in some cases including differentchemical moieties) were conjugated to the N-terminal end of the BBNbinding region (i.e., to BBN-8 or Trp⁸). BBN analogue numbers 9, 15,15i, 16, 16i and 18 were synthesized as examples of N-terminal modifiedpeptides as shown in FIG. 5.

[0075] Various tethered N-terminal (via Trp⁸) BBN analogues were alsosynthesized by SPPS as exemplified by BBN-40, BBN-41, BBN-42, BBN-43,BBN-44, BBN-45, and BBN-49 as shown in FIG. 6. In these particulartethered peptides, a Glu residue was attached to Trp⁸ followed byattachment of FmOC protected terminal amine groups separated from a-COOH group by 3-, 4-, 5-, 6-, 8- and 11-carbon chain (CH) spacers (FIG.6). These FmOC protected acids were added as the terminal step duringthe SPPS cycle. As described previously, each of the BBN analogues waspurified by reversed-phase HPLC and characterized by high resolutionMass Spectroscopy. Peptide 49 employed only glutamine as the spacergroup.

[0076] The [16]aneS₄ macrocyclic ligand was conjugated to selectedtethered BBN analogues shown in FIG. 6. The —OCH₂COOH group on the[16]aneS₄ macrocycle derivative was activated via HOBt/HBTU so that itefficiently formed an amide bond with the terminal NH₂ group on thespacer side arm (following deprotection). The corresponding [16]aneS₄tethered BBN derivatives were produced and examples of 4 of thesederivatives (i.e., BBN-22, -37, -46 and -47) are shown in FIG. 7. Aspreviously described, each [16]aneS₄ BBN derivative was purified byreversed phase HPLC and characterized by FAB Mass Spectroscopy.

[0077] B. In Vitro Binding Affinities

[0078] The binding affinities of the synthetic BBN derivatives wereassessed for GRP receptors on Swiss 3T3 cells and, in some cases, on avariety of human cancer cell lines, that express GRP receptors. TheIC₅₀'s of each derivative was determined relative to (i.e., incompetition with) ¹²⁵I-Tyr⁴-BBN (the K_(d) for ¹²⁵I-Tyr⁴-BBN for GRPreceptors in Swiss 3T3 cells is reported to be 1.6±0.4 nM) [Zueht etal., 1991]. The cell binding assay methods used to measure the IC₅₀'s isstandard and was used by techniques previously reported [Jensen et al.,1993; Cai et al., 1994; Cai et al., 1992]. The methods used fordetermining IC₅₀'s with all GRP receptor binding of GRP receptors on allcell lines was similar. The specific method used to measure IC₅₀'s onSwiss 3T3 cells is briefly described as follows:

[0079] Swiss 3T3 mouse fibroblasts are grown to confluence in 48 wellmicrotiter plates. An incubation media was prepared consisting of HEPES(11.916 g/l), NaCl (7.598 g/l), KCl (0.574 g/l), MgCl₂ (1.106 g/l), EGTA(0.380 g/l), BSA (5.0 g/l), chymostatin (0.002 g/l), soybean trypsininhibitor (0.200 g/l), and bacitracin (0.050 g/l). The growth media wasremoved, the cells were washed twice with incubation media, andincubation media was returned to the cells. ¹²⁵I-Tyr⁴-BBN (0.01 uCi) wasadded to each well in the presence of increasing concentrations of theappropriate competitive peptide. Typical concentrations of displacingpeptide ranged from 10⁻¹² to 10⁻⁵ moles of displacing ligand per well.The cells were incubated at 37° C. for forty minutes in a 95%O₂/5%CO₂humidified environment. At forty minutes post initiation of theincubation, the medium was discarded, and the cells were washed twicewith cold incubation media. The cells were harvested from the wellsfollowing incubation in a trypsin/EDTA solution for five minutes at 37°C. Subsequently, the radioactivity, per well, was determined and themaximum % total uptake of the radiolabeled peptide was determined andnormalized to 100%.

[0080] C. Results of Binding Affinity Measurements

[0081] The IC₅₀ values measured for the BBN derivatives synthesized inaccordance with this invention showed that appending a peptide sidechain and other moieties via the N-terminal BBN-8 residue (i.e., Trp⁸)produced widely varying IC₅₀ values. For example, see IC₅₀ values shownfor BBN 11, 15i, 16i, and 18 in FIGS. 5 and 8. The observations areconsistent with previous reports showing highly variable IC₅₀ valueswhen derivatizing BBN(8-13) or BBN(8-14) with a predominantly shortchain of amino acid residues [Hoffken, 1994]. In contrast, when ahydrocarbon spacer of 3- to 11-carbons was appended between BBN(7-14)and the [16]aneS₄ macrocycle, the IC₅₀'s were found to be surprisinglyrelatively constant and in the 1-5 nM range (i.e., see IC₅₀ values forBBN-22, -37, -46 and -47 as shown in FIG. 7). These data suggest thatusing relatively simple spacer groups to extend ligands some distancefrom the BBN binding region [e.g., BBN(8-14)] can produce derivativesthat maintain binding affinities in the 1-5 nmolar range.

[0082] D. Cell Binding Studies

[0083] Results illustrated in FIG. 9 show that when the RhCl₂-[16]aneS₄complex separated from Trp⁸ by only a glutamine (Glu⁷), the IC₅₀ of thisconjugate (i.e., Rh-BBN-22) was 37.5 nM. However, when a five (5) carbonspacer or an eight (8) carbon spacer was present (i.e., Rh-BBN-37 andRh-BBN-47), the IC₅₀'s remained below 5 nM as shown in FIG. 9. Thesedata demonstrate that a straight chain spacer (along with glu⁷) to movethe +1 charged Rh—S₄-chelate away from the BBN binding region willresult in a metallated BBN analogue with sufficiently high bindingaffinities to GRP receptors for in vivo tumor targeting applications.

[0084] E. ¹⁰⁵Radiolabeled BBN Analogues

[0085] The ¹⁰⁵Rh conjugates of BBN-22, BBN-37, BBN-46 and BBN-47 weresynthesized using a ¹⁰⁵Rh-chloride reagent from the Missouri UniversityResearch Reactor (MURR). This reagent was obtained as ¹⁰⁵Rh-chloride, ano-carrier-added (NCA) product, in 0.1-1M HCl. The pH of this reagentwas adjusted to 4-5 using 0.1-1.0 M NaOH dropwise and it was added toapproximately 0.1 mg of the [16]aneS₄-conjugated BBN derivatives in 0.9%aqueous NaCl and 10% ethanol. After the sample was heated at 80° C. forone hour, the ¹⁰⁵Rh-BBN analogues were purified using HPLC. In eachcase, a NCA or high specific activity product was obtained since thenon-metallated S₄-BBN conjugates eluted at a retention time well afterthe ¹⁰⁵Rh-BBN conjugates eluted. For example, the retention time of¹⁰⁵Rh-BBN-37 was 7.1 min while BBN-37 eluted at 10.5 min from aC-18-reversed phase column eluted with CH₃CN/H₂O containing 0.1% TFA asshown in FIGS. 10A-B.

EXAMPLE II Retention of ¹⁰⁵Rh-BBN Analogues in Cancer Cells

[0086] Once the radiometal has been specifically “delivered” to cancercells (e.g., employing the BBN binding moiety that specifically targetsGRP receptors on the cell surface), it is necessary that a largepercentage of the “delivered” radioactive atoms remain associated withthe cells for a period time of hours or longer to make an effectiveradiopharmaceutical for effectively treating cancer. One way to achievethis association is to internalize the radiolabeled BBN conjugateswithin the cancer cell after binding to cell surface GRP receptors.

[0087] In the past, all of the work with synthetic-BBN analogues fortreatment of cancers focused on synthesizing and evaluating antagonists[Davis et al., 1992; Hoffken, 1994; Moody et al., 1996; Coy et al.,1988; Cai et al., 1994; Moody et al., 1995; Leban et al., 1994; Cai etal., 1992]. After evaluating synthetic BBN analogues that would bepredicted to be either agonists or antagonists, applicants found thatderivatives of BBN(8-14) (i.e., those with the methionine or amidatedmethionine at BBN-14) are rapidly internalized (i.e., in less than twominutes) after binding to the cell surface GRP receptors. Severalradiolabeled BBN(8-14) analogues that were studied to determine theirinternalization and intracellular trapping efficiencies wereradioiodinated (i.e., 125I) derivatives. The results of these studiesdemonstrated that despite rapid internalization after ¹²⁵I-labeled BBNanalogue binding to GRP receptors in Swiss 3T3 cells, the ¹²⁵I wasrapidly expelled from the cells [Hoffman et al., 1997] as shown in FIG.11. Thus, these ¹²⁵I-BBN derivatives were not suitable for furtherdevelopment.

[0088] In contrast, the ¹⁰⁵Rh-BBN(8-14) derivatives that bind to GRPreceptors are not only rapidly internalized, but there is a largepercentage of the ¹⁰⁵Rh activity that remains trapped within the cellsfor hours (and in some cell lines >twenty four hours). This observationindicates that these radiometallated BBN derivatives have real utilityas radiopharmaceuticals for in vivo targeting of neoplasms expressingGRP receptors.

[0089] Experiments designed to determine the fraction of a radiotracerinternalized within cells were performed by adding excess 125I- or¹⁰⁵Rh-BBN derivatives to the cell incubation medium. After establishmentof equilibrium after a forty minute incubation, the media surroundingthe cells was removed and the cells were washed with fresh mediacontaining no radioactivity. After washing, the quantity ofradioactivity associated with the cells was determined (i.e., totalcounts per min (TCPM) of ¹²⁵I or ¹⁰⁵Rh associated with the cells). Thecells were then incubated in a 0.2M acetic acid solution (pH 2.5) whichcaused the surface proteins (incl., GRP receptors) to denature andrelease all surface bound radioactive materials. After removing thisbuffer and washing, the cells were counted again. The counts per minute(c.p.m.) associated with the cells at that point were only related tothe 125I or ¹⁰⁵Rh that remained trapped inside of the cells.

[0090] To determine intracellular retention, a similar method wasemployed. However, after washing the cells with fresh (non-radioactive)incubation media, the cells were incubated in the fresh media atdifferent time periods after washing away all extracellular 125I- or¹⁰⁵Rh-BBN analogues. After each time period, the methods used todetermine TOTAL c.p.m. and intracellular c.p.m. after washing with a0.2M acetic acid solution at pH 2.5 were the same as described above andthe percent ¹²⁵I or ¹⁰⁵Rh remaining trapped inside of the cells wascalculated. FIG. 12 is a graph of results of efflux experiments usingSwiss 3T3 cells with ¹²⁵I-Lys³-BBN. The results show that there is rapidefflux of the 125I from inside of these cells with less than 50%retained at fifteen minutes and by sixty minutes, less than 20% remainedas shown in FIG. 12.

[0091] In contrast, studies with all of the ¹⁰⁵Rh-[16]aneS₄-BBN agonistderivatives that are internalized inside of the cells showed substantialintracellular retention of ¹⁰⁵Rh by the GRP receptor expressing cells.For example, results of studies using ¹⁰⁵Rh-BBN-37 (see FIG. 9) inconjunction with Swiss 3T3 cells showed that approximately 50% of the¹⁰⁵Rh activity remains associated with the cells at sixty minutespost-washing and approximately 30% of ¹⁰⁵Rh remained inside of the cellsafter four hours as shown in FIG. 13. Note that at least 5% of the ¹⁰⁵Rhis surface bound at >sixty minutes.

[0092] The ¹⁰⁵Rh-BBN derivatives shown in FIG. 9 all have an amidatedmethionine at position BBN-14 and are expected to be agonists [Jensen etal., 1993]. Therefore, they would be predicted to rapidly internalizeafter binding to GRP receptors on the cell surface [Reile et al., 1994;Bjisterbosch et al., 1995; Smythe et al., 1991], which was confirmed byapplicants' data. Referring to FIG. 14, ¹⁰⁵Rh-BBN-61, a BBN analoguewith no amino acid at position BBN-14 (i.e., a ¹⁰⁵Rh-BBN(8-13)derivative), was synthesized and studied. This BBN analogue has a highbonding affinity (i.e., IC₅₀=30 nM). This type of derivative is expectedto be an antagonist and as such will not internalize [Jensen et al.,1993; Smythe et al., 1991]. Results of efflux studies with ¹⁰⁵Rh-BBN-61using Swiss 3T3 cells showed that immediately following washing withfresh incubation buffer (i.e., t=0), essentially all of the ¹⁰⁵Rhassociated with these cells is on the cell surface, as expected.Furthermore, after only one hour of incubation, less than 10% remainedassociated with these cells in any fashion (see FIGS. 15 and 16). Thesedata indicate that ¹⁰⁵Rh-antagonists with structures similar to the¹⁰⁵Rh-BBN agonists (i.e., those shown in FIG. 9) are not good candidatesfor development of radiopharmaceuticals since they are neither trappedin nor on the GRP receptor expressing cells to nearly the same extent asthe radiometallated BBN agonists.

EXAMPLE III Human Cancer Cell Line Studies

[0093] In vitro cell binding studies of ¹⁰⁵Rh-BBN-37 with two differenthuman cancer cell lines that express GRP receptors (i.e., the PC-3 andCF-PAC1 cell lines), which are tumor cells derived from patients withprostate CA and pancreatic CA, as shown in FIGS. 17A-B and 18A-B,respectively) were performed. Results of these studies demonstratedconsistency with ¹⁰⁵Rh-BBN-37 binding and retention studies using Swiss3T3 cells. Specifically, the binding affinity of Rh-BBN-37 was high(i.e., IC₅₀≅7 nM) with both human cancer cell lines as shown in Table 1.In addition, in all cells, the majority of the ¹⁰⁵Rh-BBN-37 wasinternalized and perhaps a major unexpected result was that theretention of the ¹⁰⁵Rh-tracer inside of the cells was significantlybetter than retention in Swiss 3T3 cells as shown in FIGS. 17 and 18.For example, it is particularly remarkable that the percentage of¹⁰⁵Rh-BBN-37 that remained associated with both the CFPAC-l and PC-3cell line was >80% at two hours after removing the extracellularactivity by washing with fresh incubation buffer (see FIGS. 17 and 18).

EXAMPLE IV In vitro Studies

[0094] Biodistribution studies were performed by intravenous (I.V.)injection of either ¹⁰⁵Rh-BBN-22 or ¹⁰⁵Rh-BBN-37 into normal mice. Inthese studies, unanesthetized CF-1 mice (15-22 g, body wt.) wereinjected I.V. via the tail vein with between one (1) to five (5) uCi(37-185 KBq) of the ¹⁰⁵Rh-labeled agent. Organs, body fluids and tissueswere excised from animals sacrificed at 30, 60 and 120 minutespost-injection (PI). The tissues were weighed, washed in saline (whenappropriate) and counted in a NaI well counter. These data were thenused to determine the percent injected dose (% ID) in an organ or fluidand the %ID per gram. The whole blood volume of each animal wasestimated to be 6.5 percent of the body weight. Results of these studiesare summarized in Tables 2 and 3.

[0095] Results from these studies showed that both the ¹⁰⁵Rh-BBN-22 and¹⁰⁵Rh-BBN-37 were cleared from the blood stream, predominantly via thekidney into the urine. Specifically, 68.4±6.6% and 62.3±5.8% of the IDwas found in urine at two hours PI of ¹⁰⁵Rh-BBN-22 and ¹⁰⁵Rh-BBN-37,respectively (see Tables 2 and 3). An unexpected finding was that the %ID of ¹⁰⁵Rh that remained deposited in the kidneys of these animals wasonly 2.4±0.6% ID and 4.6±1.3% ID at two hours PI of ¹⁰⁵Rh-BBN-22 and¹⁰⁵Rh-BBN-37 (see Tables 2 and 3). This is much less than would beexpected from previously reported data where radiometallated peptidesand small proteins have exhibited renal retention of the radiometal thatis >10% ID and usually much >10% [Duncan et al., 1997]. The reason forreduced renal retention of ¹⁰⁵Rh-BBN analogues is not known, however,this result demonstrates a substantial improvement over existingradiometallated peptides.

[0096] Biodistribution studies also demonstrated another important invivo property of these radiometallated BBN analogues. Both ¹⁰⁵Rh-BBN-22and ¹⁰⁵Rh-BBN-37 are efficiently cleared from organs and tissues that donot express GRP receptors (or those that do not have their GRP-receptorsaccessible to circulating blood). The biodistribution studies in micedemonstrated specific uptake of ¹⁰⁵Rh-BBN-22 and ¹⁰⁵Rh-BBN-37 in thepancreas while other non-excretory organs or tissues (i.e., heart,brain, lung, muscle, spleen) exhibited no uptake or retention (Tables 2and 3). Both ¹⁰⁵Rh-BBN-22 and ¹⁰⁵Rh-BBN-37 were removed from the bloodstream by both the liver and kidneys with a large fraction of the ¹⁰⁵Rhremoved by these routes being excreted into the intestines and thebladder, respectively. It is important to note that the % ID/gm in thepancreas of ¹⁰⁵Rh-BBN-22 and ¹⁰⁵Rh-BBN-37 was 3.9±1.3% and 9.9±5.4%,respectively at 2 hr, PI. Thus, the ratios of % ID/gm of ¹⁰⁵Rh-BBN-22 inthe pancreas relative to muscle and blood were 16.2 and 7.6,respectively. The ratios of % ID/gm of ¹⁰⁵Rh-BBN-37 in the pancreasrelative to muscle and blood were 25.4 and 29.1, respectively. Thesedata demonstrated selective in vivo targeting of these radiometallatedBBN analogues to cells expressing GRP receptors [Zhu et al., 1991; Qinet al., 1994] and efficient clearance from non-target tissues. If cancercells that express GRP receptors are present in the body, these resultsindicate radiometallated BBN analogues will be able to target them witha selectivity similar to the pancreatic cells.

[0097] A comparison of the pancreatic uptake and retention of¹⁰⁵Rh-BBN-22 with ¹⁰⁵Rh-BBN-37 demonstrated that ¹⁰⁵Rh-BBN-37 depositsin the pancreas with a 2-fold better efficiency than ¹⁰⁵Rh-BBN-22 (i.e.,3.6±1.2% ID and 2.3±1.0% ID) for ¹⁰⁵Rh-BBN-37 at one and two hours PI,respectively, vs. 1.2±0.5% ID and 1.0±0.1% ID for ¹⁰⁵Rh BBN-22 at oneand two hours PI). This data is consistent with the >2-fold higheruptake and retention of ¹⁰⁵Rh-BBN-37 found in the in vitro studies shownin FIG. 16.

EXAMPLE V Synthesis and in vitro Binding Measurement of Synthetic BBNConjugate Analogues Employing Amino Acid Chain Spacers

[0098] A. Synthesis

[0099] Five BBN analogues were synthesized by SPPS in which between 2 to6 amino acid spacer groups were inserted to separate a S₄-macrocyclicchelator from the N-terminal trp⁸ on BBN(8-14) (FIG. 19). Each peptidewas prepared by SPPS using an Applied Biosystems Model 432A peptidesynthesizer. After cleavage of each BBN analogue from the resin usingTrifluoracetic acid (TFA), the peptides were purified by C₁₈reversed-phase HPLC using a Vydac HS54 column and CH₃CN/H₂O containing0.1% TFA as the mobile phase. After collection of the fractioncontaining the desired BBN peptide, the solvent was evaporated. Theidentity of each BBN peptide was confirmed by FAB-mass spectrometry(Department of Chemistry—Washington University, St. Louis, Mo.).

[0100] Various amino acid sequences (in some cases containing differentR group moieties) were conjugated to the N-terminal end of the BBNbinding region (i.e., to BBN-8 or Trp⁸). BBN analogue numbers 96, 97,98, 99 and 101 were synthesized as examples of N-terminal modifiedpeptides in which the [16]aneS₄ macrocycle BFCA was separated from trp⁸on BBN(8-14) by various amino acid sequences as shown in FIG. 19.

[0101] The [16]aneS₄ macrocyclic ligand was conjugated to selectedtethered BBN analogues. The —OCH₂COOH group on the [16]andS₄ macrocyclederivative was activated via HOBt/HBTU so that it efficiently formed anamide bond with the terminal NH2 group on the spacer side arm (followingdeprotection). The corresponding [16]aneS₄ tethered BBN derivatives wereproduced and examples of 5 of these derivatives (i.e., BBN-96, 97, 98,99 and 101) are shown in FIG. 19. As previously described, each[16]aneS₄ BBN derivative was purified by reversed phase HPLC andcharacterized by FAB Mass Spectroscopy.

[0102] B. In vitro Binding Affinities

[0103] The binding affinities of the synthetic BBN derivatives wereassessed for GRP receptors on Swiss 3T3 cells, PC-3 cells and CF PAC-1cells. The IC₅₀'s of each of derivative was determined relative to(i.e., in competition with) ¹²⁵I-Tyr⁴-BBN. The cell binding assaymethods used to measure the IC₅₀'s is standard and was used bytechniques previously reported [Jensen et al., 1993; Cai et al., 1992;Cai et al., 1994]. The methods used for determining IC₅₀'s with all BBNanalogue binding to GRP repectors present on all three cell lines wassimilar. The specific method used to measure IC₅₀'s on Swiss 3T3 cellsis briefly described as follows:

[0104] Swiss 3T3 mouse fibroblasts are grown to confluence in 48 wellmicroliter plates. An incubation media was prepared consisting of HEPES(11.916 g/l), NaCl (7.598 g/l), KCl (0.574 g/l), MgCl₂(1.106 g/l), EGTA(0.380 g/l), BSA (5.0 g/l), chymostatin (0.002 g/l), soybean trypsininhibitor (0.200 g/l), and bacitracin (0.050 g/l). The growth media wasremoved, the cells were washed twice with incubation media, andincubation media was returned to the cells. ¹²⁵I-Tyr⁴-BBN (0.01 μCi) wasadded to each well in the presence of increasing concentrations of theappropriate competitive peptide. Typical concentrations of displacingpeptide ranged from 10⁻¹² to 10⁻⁵ moles of displacing ligand per well.The cells were incubated at 37° C. for forty minutes in a 95% O₂/5% CO₂humidified environment. At forty minutes post initiation of theincubation, the medium was discarded, and the cells were washed twicewith cold incubation media. The cells were harvested from the wellsfollowing incubation in a trypsin/EDTA solution for five minutes at 37°C. Subsequently, the radioactivity, per well, was determined and themaximum % total uptake of the radiolabeled peptide was determined andnormalized to 100%. A similar procedure was used in performing cellbinding assays with both the PC-3 and CF_(a)-PAC-1 human cancer celllines.

[0105] C. Results of Binding Affinity Measurements

[0106] The IC₅₀ values measured for the BBN derivatives synthesized inaccordance with this invention showed that appending a chelator viaamino acid chain spacer groups via the N-terminal BBN-8 residue (i.e.,Trp⁸) produced a variation of IC₅₀ values. For example, see IC₅₀ valuesshown for BBN 96, 97, 98 and 101 in FIG. 19. The observations areconsistent with previous reports showing variable IC₅₀ values whenderivatizing BBH(8-13) with a predominantly short chain of amino acidresidues [Hoffken, 1994]. When the amino acid spacer groups used inBBN-98, 99 and 101 were appended between BBN(7-14) and the [16]aneS₄macrocyle, the IC₅₀'s were found to be surprisingly constant and in the1-6 nM range for all three cell lines (i.e., see IC₅₀ values shown inFIG. 19). These data suggest that using relatively simple spacer groupscomposed entirely of selected amino acid sequences to extend ligandssome distance from the BBN region [e.g., BBN(8-14) can producederivatives that maintain binding affinities in the 1-6 nmolar range.

[0107] D. Cell Binding Studies with Rh-BBN-Conjugates

[0108] Results illustrated in FIG. 20 show that when the correspondingRhCl₂ [16]aneS₄ complex was separated from Trp⁸ on BBH(8-14) by the fourdifferent amino acid spacer groups (see FIG. 20), the IC₅₀'s of all fouranalogues (i.e., BBN-97, -98, -99, -101) were between 0.73 and 5.29nmolar with GRP receptors on the PC-3 and CF PAC-1 cell lines. TheIC₅₀'s for these same Rh-BBN conjugates were somewhat higher with theSwiss 3T3 cell line (FIG. 20). These data demonstrate that amino acidchain with spacer groups used to move the +1 charged Rh—S₄-chelate awayfrom the BBN binding region will result in a metallated BBN analoguewith sufficiently high binding affinities to GRP receptors for in vivotumor targeting applications.

EXAMPLE VI Synthesis and in vitro Binding Assessment of a^(99m)Tc-Labeled Synthetic BBN Analogue

[0109] A. Synthesis

[0110] Several tetradentate chelating frameworks have been used to formstable ^(99m)Tc or ¹⁸⁸Re labeled peptide and protein conjugates[Eckelman, 1995; Li et al., 1996b; Parker, 1990; Lister-James et al.,1997]. Many of these ligand systems contain at least one thiol (—SH)donor group to maximize rates of formation and stability (both in vitroand in vivo) of the resultant Tc(V) or Re(V) complexes [Parker, 1990;Eckelman, 1995]. Results from a recent report indicates that thebifunctional chelating agent (BFCA)(dimethylglycyl-L-seryl-L-cyteinyl-glycinamide (N₃S-BFCA) is capable offorming a well-defined complex with ReO⁺³ and TcO⁺³ [Wong et al., 1997].Since this ligand framework can be synthesized by SPPS techniques, thisN₃S-BFCA was selected for use in forming of Tc-99m-BBN-analogueconjugates. Three different N₃S-BFCA conjugates of BBN(7-14) weresynthesized (BBN-120, -121 and -122) as shown in FIG. 21 by SPPS.BBN-120, BBN-121 and BBN-122 represent a series of analogues where theN₃S-BFCA is separated from the BBN(7-14) sequence by a 3, 5 and 8 carbonspacer groups (FIG. 21). Each peptide was synthesized and purified usingthe SPPS and chromatographic procedures outlined in Example 1. The thiolgroup on cystein was protected using the ACM group, which is not cleavedduring cleavage of these BBN-conjugates from the resin using TFA. Theidentity of BBN-120, -121 and -122 was confirmed by FAB massspectrometry. Synthesis and purification of the N₃S-BFCA could also bereadily accomplished using SPPS methods, followed by HPLC purification(see Example 1). The ACM group was used to protect the thiol group oncysteine during synthesis and cleavage from the resin.

[0111] B. In vitro Binding Affinities

[0112] Synthesis of ^(99m)Tc-BBN-122 (FIG. 22) was prepared by twomethods [i.e., (1) by transchelation of ^(99m)TcO⁺³ from^(99m)Tc-gluconate or (2) by formation of the “preformed” ^(99m)Tc-BFCAcomplex followed by —COOH activation with tetrafluorophenyl andsubsequent reaction with the C₅-carbon spacer group appended toBBN(7-14)]. In both cases, the ^(99m)Tc-labeled peptide formed is shownin FIG. 22. The structure of this Tc-BBN-122 conjugate was determined byusing non-radioactive Re(the chemical congener of Tc). In these studies,the “preformed” ReO⁺³ complex with the N₃S-BFCA was prepared byreduction of ReO₄; with SnCl₂ in the presence of excess N₃S-BFCAdissolved in sodium phosphate buffered water at pH 6-6.5 by a methodpreviously published [Wong et al., 1997]. After purification of theReO—N₃S-BFCA complex, the structure of this chelate was shown (byMass-Spect) to be identical to that previously reported [Wong et al.,1997].

[0113] The ReO—N₃—S—BFCA complex was converted to the activatedtrifluorophenyl (TFP) ester by adding 10 mg of the complex to 6 mg (dry)EDC and the 50 μl of TFP. After the solution was vortexed for oneminute, CH₃CN was added until disappearance of cloudiness. The solutionwas incubated for one hour at RT and purified by reversed-phase HPLC. Toprepare the ReO—N₃S-BFCA complex BBN-122 conjugate (FIG. 22), one μl ofthe HPLC fraction containing the ReO—N₃S—BFCA complex was added to asolution containing 1 mg of the C₈-tethered BBN(7-14) peptide in 0.2 NNaHCO₃ at pH 9.0. After incubation of this solution for one hour at RT,the sample was analyzed and purified by reversed-phase HPLC. The yieldof Re-BBN-122 was approximately 30-35%.

[0114] The method for preparation of the corresponding ^(99m)Tc-BBN-122conjugate, using the “preformed” ^(99m)TcO—N₃S-BFCA complex, was thesame as described above with the “preformed” ReO—N₃S-BFCA complex. Inthis case, ^(99m)TcO₄, from a ⁹⁹Mo/^(99m)Tc generator, was reduced withan aqueous saturated stannous tartrate solution in the presence ofexcess N₃S-BFCA. The yields of the ^(99m)Tc-BBN-122 product using this“preformed” method were approximately 30-40%. Reversed phase HPLCanalysis of the 99mTc-BBN-122, using the same gradient elution program¹as used for analysis of the Re-BBN-122 conjugate, showed that

[0115] both the ^(99m)Tc-BBN-122 and ¹⁸⁸Re-BBN-122 had the sameretention time (i.e., 14.2-14.4 min) (See FIG. 22). This provides strongevidence that the structure of both the ^(99m)Tc-BBN-122 and Re-BBN-122are identical.

[0116] The binding affinities of BBN-122 and Re-BBN-122 were assessedfor GRP receptors on Swiss 3T3 cells, PC-3 cells and CFPAC-1 cells thatexpress GRP receptors. The IC₅₀'s of each derivative was determinedrelative to (i.e., in competition with) ¹²⁵I-Tyr⁴-BBN (the K_(d) for¹²⁵I-Tyr⁴-BBN for GRP receptors in Swiss 3T3 cells is reported to be1.6±0.4nM) [Zhu et al., 1991]. The cell binding assay methods used tomeasure the IC₅₀'s is standard and was used by techniques previouslyreported [Leban et al., 1994; Cai et al., 1994; Cai et al., 1992]. Themethods used for determining IC₅₀'s with all GRP receptor binding of GRPreceptors on all cell lines was similar and has been describedpreviously for the other BBN-analogues and Rh-BBN analogues described inthis document.

[0117] C. Results of Binding Affinity Measurements

[0118] The IC₅₀ values measured for BBN-122 and Re-BBN-122 synthesizedin accordance with this invention showed that appending an Time(minutes) % A/ % B  0 95/5  25 30/70 35 95/5 

[0119] 8-carbon hydrocarbon chain spacer linked to the N₂S₁-BFCA and thecorresponding Re complex (i.e., Trp⁸) produced BBN conjugates with IC₅₀values in a 1-5 nmolar range (See Table A). When ^(99m)Tc-BBN-122 wasincubated with these same cells, it was shown that ≧nmolarconcentrations of BBN displaced this ^(99m)Tc conjugate by >90%. Thisresult demonstrates that ^(99m)Tc-BBN-122 has high and specific bindingaffinity for GRP receptors. These data suggest that using relativelysimple spacer groups to extend the N₃S ligand framework and thecorresponding Tc— or Re—N₃S₁, complexes some distance from the BBNbinding region can produce derivatives that maintain binding affinitiesin the 1-5 nmolar range.

[0120] TABLE A.

[0121] Summary of IC₅₀ values for GRP receptor binding for thenon-metallated BBN-122 conjugate or the Re-BBN-122 conjugate in two celllines (PC-3 and CF-PAC-1 cell lines that express GRP receptors). TheIC₅₀ values were measured using cell binding assays relative to¹²⁵I-Tyr⁴-BBN. IC₅₀ (nmolar) Conjugate PC-3 CF-PAC1 BBN-122 3.59 ± 0.75(n = 6)  5.58 ± 1.92 (n = 14) Re-BBN-122 1.23 ± 0.56 (n = 12) 1.47 ±0.11 (n = 6) 

EXAMPLE VII Retention of 99m Tc-BBN-122 in Human Cancer Cells PC-3 andCF-PAC-1 Cells

[0122] Once the radiometal has been specifically “delivered” to cancercells (e.g., employing the BBN binding moiety that specifically targetsGRP receptors on the cell surface), it is necessary that a largepercentage of the “delivered” radioactive atoms remain associated withthe cells for a period time of hours or longer to make an effectiveradiopharmaceutical for effectively treating cancer. One way to achievethis association is to internalize the radiolabeled BBN conjugateswithin the cancer cell after binding to cell surface GRP receptors.

[0123] Experiments designed to determine the fraction ^(99m)Tc-BBN-122internalized within cells were performed by the same method previouslydescribed for ¹⁰⁵Rh-BBN-37. Briefly, excess ^(99m)Tc-BBN-122 was addedto PC-3 or CFPAC-1 cell incubation media and allowed to establishequilibrium after a forty minute incubation. The media surrounding thecells was removed and the cells were washed with fresh media containingno radioactivity. After washing, the quantity of radioactivityassociated with the cells was determined (i.e., total counts per min^(99m)Tc associated with cells). The PC-3 and CFPAC-1 cells were thenincubated in a 0.2M acetic acid solution (pH 2.5) which caused thesurface proteins (including GRP receptors) to denature and release allsurface bound radioactive materials. After removing this buffer andwashing, the cells were counted again. The counts per minute (c.p.m.)associated with the cells at that point were only related to the^(99m)Tc that remained trapped inside of the PC-3 or CFPAC-1 cells.

[0124] To determine intracellular retention of ^(99m)Tc activity, asimilar method was employed. However, after washing the cells with fresh(non-radioactive) incubation media, the cells were incubated in thefresh media at different time period after washing away allextracellular ^(99m)Tc-BBN-122. After each time interval, the methodsused to determine total c.p.m. and intracellular c.p.m. by washing witha 0.2M acetic acid solution at pH 2.5.

[0125] Studies with the 99mTc-BBN-122 agonist show that it isinternalized inside of the PC-3 and CFPAC-1 cells (FIGS. 23-26) and thatsubstantial intracellular retention of ^(99m)Tc by the GRP receptorexpressing cells occurs. For example, results of studies using^(99m)Tc-BBN-122 in conjunction with PC-3 cells showed a high rate ofinternalization (FIG. 23) and that approximately 75% of the ^(99m)Tcactivity remains associated with the cells at ninety minutespost-washing (FIG. 25). Almost all of this ^(99m)Tc cell-associatedactivity is inside of the PC-3 cells. Similar results were also foundwith the CFPAC 1 cells where there is also a high rate of^(99m)Tc-BBN-122 internalization (FIG. 24) and relatively slow efflux of^(99m)Tc from the cells (i.e., 50-60% retention at 120 min post-washing(FIG. 26).

[0126] The ^(99m)Tc-BBN-122 peptide conjugate shown in FIG. 22 has anamidated methionine at position BBN-14 and is expected to be an agonists[Jensen et al., 1993]. Therefore, it would be predicted to rapidlyinternalize after binding to GRP receptors on the cell surface[Bjisterbosch et al., 1995; Smythe et al., 1991], which is confirmed byapplicants' data in FIG. 23-26.

EXAMPLE VIII In vivo Studies

[0127] Biodistribution studies were performed by intravenous (I.V.)injection of ^(99m)Tc-BBN-122 into normal mice. In these studies,unanesthetized CF-1 mice (15-22 g, body wt.) were injected I.V. via thetail vein with between one (1) to five (5) μCi (37-185 KBq) of^(99m)Tc-BBN-122. Organs, body fluids and tissues were excised fromanimals sacrificed at 0.5, 1, 4 and 24 hours post-injection (PI). Thetissues were weighed, washed in saline (when appropriate) and counted ina NaI well counter. These data were then used to determine the percentinjected dose (% ID) in an organ or fluid and the % ID) per gram. Thewhole blood volume of each animal was estimated to be 6.5 percent of thebody weight. Results of these studies are summarized in Tables B and C.

[0128] Results from these studies showed that 99mTc-BBN-122 is clearedfrom the blood stream predominantly via the hepatobiliary pathwayshaving about 35% of the ^(99m)Tc-activity cleared via the kidney intothe urine. Specifically, 33.79±1.76% of the ID was found in urine at onehour PI (Table B). The retention of ^(99m)Tc activity in the kidneys andliver is very low (Table B). This is much less than would be expectedfrom previously reported data where radiometallated peptides and smallproteins have exhibited renal retention of the radiometal that is >10%ID and usually much >10% [Duncan et al., 1997]. The reason for reducedrenal retention of ^(99m)Tc-BBN-122 is not known, however, this resultdemonstrates a substantial improvement over existing radiometallatedpeptides.

[0129] Biodistribution studies also demonstrated another important invivo property of ^(99m)Tc-BBN-122 in that it is efficiently cleared fromorgans and tissues that do not express GRP receptors (or those that donot have their GRP-receptors accessible to circulating blood). Thebiodistribution studies in mice demonstrated specific uptake of^(99m)Tc-BBN-122 in the pancreas while other non-excretory organs ortissues (i.e., heart, brain, lung, muscle, spleen) exhibited no uptakeor retention. ^(99m)Tc-BBN-122 is removed from the blood stream by boththe liver and kidneys with a large fraction of the 99mTc removed bythese routes being excreted into the intestines and the bladder,respectively. It is important to note that the % ID/gm in the pancreasof ^(99m)Tc-BBN-122 is 12.63%/gm at 1 hour and drops to only 5.05% atthe 4 hour PI (Table C). Thus, the ratios of % ID/gm of ^(99m)Tc-BBN-122in the pancreas relative to muscle and blood were 92.2 and 14.78 at 4hour PI, respectively. These data demonstrated selective in vivotargeting of this ^(99m)Tc-labeled BBN analogue to cells expressing GRPreceptors [Zhu et al., 1991; Qin et al., 1994] and efficient clearancefrom non-target tissues. If cancer cells that express GRP receptors arepresent in the body, these results indicate 99mTc-BBN analogues will beable to target them with a selectivity similar to the pancreatic cells.TABLE B Biodistribution of ^(99m)Tc-BBN-122 in normal CF-1 mice at 0.5,1, 4 and 24 hr post-IV injection. Results expressed as % ID/organ %Injected Dose/Organ^(a) Organ^(c) 30 min 1 hr 4 hr 24 hr Blood^(d)  3.52± 2.16  1.08 ± 0.34  0.59 ± 0.24 0.12 ± 0.01 Liver  4.53 ± 0.93  4.77 ±1.40  1.49 ± 0.32 0.32 ± 0.06 Stomach  2.31 ± 0.45  1.61 ± 0.81  1.75 ±0.20 0.30 ± 0.06 Lg. Intestine^(b)  2.84 ± 0.32 24.17 ± 7.91 23.85 ±7.02 0.61 ± 0.14 Sm. Intestine^(b) 43.87 ± 1.51 23.91 ± 9.08  5.87 ±7.09 0.42 ± 0.06 Kidneys^(b)  1.49 ± 0.19  1.15 ± 0.10  0.55 ± 0.06 0.20± 0.01 Urine^(b) 26.78 ± 1.05 33.79 ± 1.76 ˜35 ˜35 Muscle  0.02 ± 0.01 0.01 ± 0.00  0.01 ± 0.01 0.01 ± 0.01 Pancreas  5.30 ± 0.63  3.20 ± 0.83 1.21 ± 0.13 0.42 ± 0.17

[0130] TABLE C Biodistribution of ^(99m)Tc-BBN-122 in normal CF-1 miceat 0.5, 1, 4 and 24 hr post I.V. injection. Results expressed as %ID/gm. % Injected Dose/gm^(a) Organ 30 min 1 hr 4 hr 24 hr Blood^(b)2.00 ± 1.28 0.63 ± 0.19 0.34 ± 0.11 0.08 ± 0.00 Liver 2.70 ± 0.41 3.14 ±0.81 0.96 ± 0.20 0.22 ± 0.05 Kidneys 3.99 ± 0.76 3.10 ± 0.31 1.58 ± 0.150.64 ± 0.08 Muscle 0.23 ± 0.08 0.13 ± 0.02 0.05 ± 0.01 0.01 ± 0.01Pancreas 16.89 ± 0.95  12.63 ± 1.87  5.05 ± 0.42 1.79 ± 0.71 P/Bl andP/M Uptake Ratios Pancreas/  8.42 19.76 14.78 20.99 Blood Pancreas/73.16 93.42 92.25 142.76  Muscle

[0131] TABLE D Biodistribution of ^(99m)Tc-BBN-122 in PC-3 tumor bearingSCID mice at 1, 4 and 24 hr post-I.V. injection. Results expressed as %ID/organ. Tumor Line: PC-3 % ID per Organ^(a) Organ^(c) 1 hr 4 hr 24 hrBlood^(b) 1.16 ± 0.27 0.47 ± 0.06 0.26 ± 0.05 Liver 1.74 ± 0.64 0.72 ±0.10 0.29 ± 0.05 Stomach 0.43 ± 0.18 0.29 ± 0.22 0.08 ± 0.02 Lg.Intestine  9.18 ± 19.42 42.55 ± 8.74  0.64 ± 0.17 Sm. Intestine 46.55 ±16.16 2.13 ± 0.76 0.31 ± 0.04 Kidneys 1.16 ± 0.20 0.60 ± 0.06 0.16 ±0.01 Urine^(d) 32.05 ± 12.78 ˜35 ˜35 Muscle 0.01 ± 0.00 0.00 ± 0.00 0.00± 0.00 Pancreas 1.69 ± 0.61 1.05 ± 0.13 0.34 ± 0.08 Tumor 1.00 ± 0.780.49 ± 0.08 0.49 ± 0.25

[0132] TABLE E Biodistribution of ^(99m)Tc-BBN-122 in PC-3 tumor bearingSCID mice at 1, 4 and 24 hr post-I.V. injection. Results expressed as %ID/Gm. Tumor Line: PC-3 % ID per gm^(a) Organ 1 hr 4 hr 24 hr Blood^(b)0.97 ± 0.26 0.31 ± 0.03 0.18 ± 0.04 Liver 2.07 ± 0.88 0.64 ± 0.05 0.26 ±0.04 Kidneys 4.80 ± 1.33 2.23 ± 0.35 0.60 ± 0.04 Muscle 0.18 ± 0.12 0.06± 0.03 0.05 ± 0.04 Pancreas 10.34 ± 3.38  5.08 ± 1.12 1.47 ± 0.23 Tumor2.07 ± 0.50 1.75 ± 0.61 1.28 ± 0.22 T/Bl, T/M, P/Bl and P/M UptakeRatios Tumor/Blood  2.13  5.52  6.79 Tumor/Muscle 11.44 25.38 21.62Pancreas/Blood 10.64 15.96  7.81 Pancreas/ 57.14 73.40 24.87 Muscle

[0133] The invention has been described in an illustrative manner, andit is to be understood that the terminology which has been used isintended to be in the nature of words of description rather than oflimitation.

[0134] Obviously, many modifications and variations of the presentinvention are possible in light of the above teachings. It is,therefore, to be understood that within the scope of the appended claimsthe invention may be practiced otherwise than as specifically describe.

[0135] Throughout this application, various publications are referencedby citation and number. Full citations for the publication are listedbelow. the disclosure of these publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art to which this invention pertains.

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[0177] TABLE 2 (% Dose) Complex ¹⁰⁵Rh-Peptide22 ¹⁰⁵Rh-Peptide22¹⁰⁵Rh-Peptide22 Organ 30 min 1 hr 2 hr (% Dose) n-9 n-9 n-9 Brain 0.08 ±0.04 ± 0.06 ± 0.02 0.01 0.09 Blood 4.48 ± 1.86 ± 0.99 ± 1.24 0.38 0.24Heart 0.13 ± 0.08 ± 0.04 ± 0.03 0.03 0.04 Lung 0.25 ± 0.20 ± 0.15 ± 0.080.09 0.09 Liver 7.97 ± 8.51 ± 8.57 ± 2.85 2.33 2.04 Spleen 0.07 ± 0.09 ±0.05 ± 0.03 0.08 0.01 Stomach 1.11 ± 0.59 ± 0.30 ± 0.76 0.21 0.16 LargeIntestine 0.73 ± 3.21 ± 8.91 ± 0.16 3.38 3.79 Small Intestine 6.29 ±6.98 ± 3.48 ± 1.87 1.87 1.78 Kidneys 4.25 ± 3.25 ± 2.44 ± 1.33 0.60 0.64Bladder 44.66 ± 62.88 ± 68.41 ± 7.29 3.84 6.63 Muscle 0.06 ± 0.03 ± 0.01± 0.03 0.03 0.01 Pancreas 0.95 ± 1.15 ± 1.01 ± 0.46 0.49 0.14 Carcass32.90 ± 12.62 ± 6.37 ± 6.61 4.77 1.17 (% Dose/Gm) Complex¹⁰⁵Rh-Peptide22 ¹⁰⁵Rh-Peptide22 ¹⁰⁵Rh-Peptide22 Organ 30 min 1 hr 2 hr(% D/GM) n-9 n-9 n-9 Brain 0.21 ± 0.14 ± 0.16 ± 0.07 0.08 0.28 Blood2.22 ± 1.02 ± 0.51 ± 0.40 0.22 0.11 Heart 0.92 ± 0.64 ± 0.38 ± 0.25 0.200.33 Lung 1.44 ± 1.24 ± 0.92 ± 0.33 0.54 0.69 Liver 4.33 ± 5.18 ± 5.17 ±1.52 1.52 1.12 Spleen 0.86 ± 1.10 ± 0.84 ± 0.38 0.65 0.53 Stomach 2.46 ±1.53 ± 0.71 ± 1.65 0.74 0.33 Large Intestine 0.78 ± 4.42 ± 10.10 ± 0.194.62 4.58 Small Intestine 4.73 ± 5.84 ± 2.86 ± 1.47 1.81 1.47 Kidneys7.57 ± 6.70 ± 4.60 ± 1.49 0.75 0.83 Muscle 0.53 ± 0.61 ± 0.24 ± 0.320.97 0.24 Pancreas 3.12 ± 4.31 ± 3.88 ± 0.99 1.98 1.25

[0178] TABLE 3 (% Dose) Complex ¹⁰⁵Rh-Pept37 ¹⁰⁵Rh-Pept37 ¹⁰⁵Rh-Pept37Organ 30 min 1 hr 2 hr (% Dose) n-5 n-9 n-7 Brain 0.03 ± 0.07 ± 0.03 ±0.01 0.11 0.03 Blood 3.09 ± 1.46 ± 0.66 ± 0.54 0.62 0.26 Heart 0.12 ±0.05 ± 0.04 ± 0.03 0.03 0.02 Lung 0.26 ± 0.12 ± 0.08 ± 0.09 0.07 0.11Liver 13.04 ± 13.00 ± 10.12 ± 1.93 3.59 1.86 Spleen 0.21 ± 0.16 ± 0.10 ±0.13 0.08 0.04 Stomach 0.80 ± 0.65 ± 0.83 ± 0.34 0.52 0.96 LargeIntestine 2.05 ± 2.96 8.07 0.69 1.67 2.25 Small Intestine 8.44 ± 11.38 ±5.04 ± 1.89 3.02 2.27 Kidneys 7.82 ± 6.04 ± 4.57 ± 2.52 1.68 1.29Bladder 39.65 ± 51.82 ± 62.32 ± 7.21 7.53 5.78 Muscle 0.06 ± 0.02 ± 0.02± 0.03 0.01 0.02 Pancreas 2.73 ± 3.63 ± 2.25 ± 1.14 1.22 1.02 Carcasss24.35 ± 9.81 ± 6.37 ± 7.69 2.91 1.73 (% Dose/Gm) Complex ¹⁰⁵Rh-Pept37¹⁰⁵Rh-Pept37 ¹⁰⁵Rh-Pept37 Organ 30 min 1 hr 2 hr (% D/GM) n-5 n-9 n-7Brain 0.10 ± 0.26 ± 0.10 ± 0.05 0.41 0.09 Blood 1.60 ± 0.72 ± 0.34 ±0.30 0.31 0.15 Heart 0.92 ± 0.38 ± 0.28 ± 0.26 0.21 0.17 Lung 1.52 ±0.76 ± 0.46 ± 0.48 0.47 0.50 Liver 7.31 ± 7.65 ± 6.30 ± 1.15 1.29 1.73Spleen 2.18 ± 1.59 ± 1.05 ± 1.17 0.71 0.44 Stomach 1.53 ± 1.63 ± 2.18 ±0.67 1.17 2.35 Large Intestine 2.46 ± 3.80 ± 11.84 ± 0.70 2.42 4.39Small Intestine 5.69 ± 7.85 ± 3.81 ± 1.26 1.87 2.01 Kidneys 14.28 ±11.21 ± 8.39 ± 2.84 3.68 2.36 Muscle 0.73 ± 0.20 ± 0.39 ± 0.39 0.14 0.38Pancreas 14.02 ± 15.54 ± 9.91 ± 3.23 6.21 5.35

What is claimed is:
 1. A compound for use as a therapeutic or diagnosticradiopharmaceutical, said compound comprising a group capable ofcomplexing a metal attached to a moiety capable of binding to a gastrinreleasing peptide (GRP) receptor.
 2. A compound as set forth in claim 1,wherein said moiety capable of binding to a gastrin releasing peptidereceptor is a gastrin releasing peptide receptor agonist.
 3. A compoundas set forth in claim 2, wherein said gastrin releasing peptide receptoragonist includes a bombesin agonist binding moiety.
 4. A compound as setforth in claim 1, wherein said group capable of complexing a metalincludes a chelating group.
 5. A compound as set forth in claim 4,wherein said chelating group is attached to said bombesin agonistbinding moiety by a spacer group.
 6. A compound as set forth in claim 5having a structure of the formula: X—Y—B wherein X is said group capableof complexing a metal; Y is said spacer group or covalent bond; and B isa bombesin agonist binding moiety.
 7. A compound as set forth in claim6, wherein Y is a C₁-C₁₀ hydrocarbon chain.
 8. A compound as set forthin claim 7, wherein Y is a C₃-C₉ hydrocarbon chain.
 9. A compound as setforth in claim 6, wherein Y includes at least one amino acid residue.10. A compound as set forth in claim 9, wherein Y includes a L-Glnresidue at the BBN-7 position.
 11. A compound as set forth in claim 5,wherein said chelating group is attached to said bombesin agonistbinding moiety at the N-terminal end of a peptide bombesin bindingmoiety.
 12. A compound as set forth in claim 11, wherein said chelatinggroup is attached to said peptide bombesin agonist binding moiety atamino acid residue 8 (trp⁸) of said bombesin agonist binding moiety. 13.A compound as set forth in claim 11, wherein said spacer is attached tosaid bombesin agonist binding moiety at the N-terminal end of saidbombesin agonist binding moiety.
 14. A compound as set forth in claim13, wherein said spacer is attached to said bombesin agonist bindingmoiety at amino acid residue 8 (D⁻ or L⁻ trp⁸) of said bombesin agonistbinding moiety.
 15. A compound as set forth in claim 1, wherein saidmetal is a diagnostically or therapeutically useful metal.
 16. Acompound as set forth in claim 15, wherein said transition metal is ametallic radioisotope selected form the group including γ and β emittingisotopes.
 17. A compound as set forth in claim 16, wherein said metallicradioisotope is a radionuclide selected from the group including ¹⁸⁶Re,¹⁸⁸Re, ^(99m)Tc, ¹⁰⁵Rh, ¹⁹⁹Au, ¹⁵³Sm, ¹⁶⁶Ho and ⁹⁰Y.
 18. A method as setforth in claim 16, wherein said metallic isotope includes oxides andnitrides thereof.
 19. A compound as set forth in claim 4 wherein saidchelating agent includes a multidentate chelating structure capable offorming a highly stable complex with metals via coordinating atoms. 20.A compound as set forth in claim 19, wherein said chelating structureincludes coordinating atoms S, N, O, or P.
 21. A compound as set forthin claim 19, wherein said chelating structure is a macrocyclic compoundincluding coordinating atoms.
 22. A compound as set forth in claim 19,wherein said chelating structure is a S₄ chelator.
 23. A compound as setforth in claim 19, wherein said compound is a N₄ chelator.
 24. Acompound as set forth in claim 19, wherein said compound is a N₂S₂chelator.
 25. A compound as set forth in claim 19, wherein said compoundis a NS₃ chelator.
 26. A compound as set forth in claim 19, wherein saidcompound is a N₃S chelator.
 27. A compound as set forth in claim 3,wherein said compound has a binding affinity for the gastrin releasingpeptide receptor that is approximately equal to or greater than that ofnative bombesin.
 28. A method for treating a subject having a neoplasticdisease, said method comprising the steps of. administering to thesubject an effective amount of a pharmaceutical comprising a metalcomplexed with a chelating group attached to a moiety capable ofspecifically binding to a gastrin releasing peptide receptor.
 29. Amethod as set forth in claim 28, wherein the moiety capable of bindingto a gastrin releasing peptide receptor is a gastrin releasing peptidereceptor agonist.
 30. A method as set forth in claim 28 furtherincluding the step of contacting the radiopharmaceutical with aneoplastic cell.
 31. A method as set forth in claim 30, wherein saidcontacting step is further defined as binding the radiopharmaceutical togastrin releasing peptide receptors expressed on the neoplastic cell.32. A method as set forth in claim 28 further including the step ofinternalizing the compound into the neoplastic cell.
 33. A method as setforth in claim 28 further including the step of retaining the compoundwithin the neoplastic cell for a period of time sufficient to initiatedeath of the neoplastic cell or provide a diagnostic image of thetumors.
 34. A method as set forth in claim 29, wherein the gastrinreleasing peptide receptor agonist includes a bombesin agonist bindingmoiety.
 35. A method as set forth in claim 34, wherein the chelatedmetal is attached to the bombesin agonist binding moiety by a spacergroup.
 36. A method as set forth in claim 34, wherein the compound hasthe structure of the formula: X—Y—B wherein X is the group capable ofbinding a metal; Y is the spacer group or a covalent bond and B is abombesin agonist binding moiety.
 37. A method forth in claim 36, whereinY is a C₁-C₁₀ hydrocarbon chain.
 38. A method as set forth in claim 37,wherein Y is a C₃-C₉ hydrocarbon chain.
 39. A method as set forth inclaim 36, wherein Y includes at least one amino acid residue.
 40. Amethod set forth in claim 39, wherein Y includes a L-Gln residue at theBBN-7 position.
 41. A method as set forth in claim 36, wherein thechelating group is attached to peptide bombesin agonist binding moietyat the N-terminal end of the bombesin agonist binding moiety.
 42. Amethod as set forth in claim 41, wherein the chelating group is attachedto the bombesin agonist binding moiety to amino acid residue 8 (D⁻ or L⁻trp⁸) of the bombesin agonist binding moiety.
 43. A method as set forthin claim 41, wherein the spacer is attached to the bombesin agonistbinding moiety at the N-terminal end of the bombesin agonist bindingmoiety.
 44. A method as set forth in claim 28, wherein the transitionmetal is a diagnostically or therapeutically useful radioactive metal.45. A method as set forth in claim 44, wherein the metal is a metallicradioisotope selected form the group including γ and β emittingisotopes.
 46. A method as set forth in claim 45, wherein the metallicradioisotope is a radionuclide selected from the group including ¹⁸⁶Re,¹⁸⁸Re, ^(99m)Tc, ¹⁰⁵I, ¹⁹⁹Au, ¹⁵³Sm, ¹⁶⁶Ho and ⁹⁰Y.
 47. A method as setforth in claim 46, wherein said metallic radioisotope includes oxidesand nitrides thereof.
 48. A compound as set forth in claim 28 whereinsaid chelating agent includes a multidentate chelating structure capableof forming a highly stable complex with metals via coordinating atoms.49. A method as set forth in claim 48, wherein said chelating structureincludes coordinating a toms S, N, O, or P.
 50. A method as set forth inclaim 48, wherein the chelating structure is a macrocyclic compoundincluding coordinating atoms.
 51. A method as set forth in claim 48,wherein said compound is a S₄ chelator.
 52. A method as set forth inclaim 48, wherein said compound is a N₄ chelator.
 53. A method as setforth in claim 48, wherein said compound is a N₂S₂ chelator.
 54. Amethod as set forth in claim 48, wherein said compound is a NS₃chelator.
 55. A method as set forth in claim 48, wherein said compoundis a N₃S chelator.
 56. A method as set forth in claim 28, wherein thecompound has a binding affinity for the gastrin releasing peptidereceptor that is approximately equal to or greater than that of nativebombesin.
 57. A method of forming a therapeutic or diagnostic compoundcomprising the step of reacting a metal complexed with a chelating groupwith a moiety capable of agonistic binding a gastrin releasing peptidereceptor.
 58. A method of forming a therapeutic or diagnostic compoundcomprising a step of reacting a metal with a chelating group alreadycovalently attached to a moiety capable of agonistic binding a gastrinreleasing peptide receptor.
 59. A method as set forth in claim 57,wherein the moiety capable of binding to a gastrin releasing peptidereceptor is a gastrin releasing peptide receptor agonist.
 60. A methodas set forth in claim 57, wherein the gastrin releasing peptide receptoragonist includes a bombesin agonist binding moiety.
 61. A method as setforth in claim 60, wherein the chelated metal is attached to thebombesin agonist binding moiety by a spacer group.
 62. A method as setforth in claim 60, wherein the compound has the structure of theformula: X—Y—B wherein X is the group capable of binding a metal; Y isthe spacer group or a covalent bond and B is a bombesin binding moiety.63. A method forth in claim 62, wherein Y is a C₁-C₁₀ hydrocarbon chain.64. A method as set forth in claim 63, wherein Y is a C₃-C₉ hydrocarbonchain.
 65. A method as set forth in claim 60, wherein Y includes atleast one amino acid residue.
 66. A method as set forth in claim 65,wherein Y includes a L-Gln residue at the BBN-7 position.
 67. A methodas set forth in claim 62, wherein the chelating group is attached to thebombesin agonist binding moiety at the N-terminal end of the bombesinagonist binding moiety.
 68. A method as set forth in claim 60, whereinthe chelating group is attached to the bombesin agonist binding moietyat amino acid residue 8 (D⁻ or L⁻ trp⁸) of the bombesin molecule orderivative thereof.
 69. A method as set forth in claim 60, wherein thespacer is attached to the bombesin agonist binding moiety at theN-terminal end of the bombesin agonist binding moiety.
 70. A method asset forth in claim 60, wherein the spacer is attached to the bombesinagonist binding moiety at amino acid residue 8 (D⁻ or L⁻ trp⁸) of thebombesin agonist binding moiety.
 71. A method as set forth in claim 57,wherein the metal is a diagnostically or therapeutically usefulradioactive metal.
 72. A method as set forth in claim 71, wherein themetal is a metallic radioisotope selected form the group including γ andβ emitting isotopes.
 73. A method as set forth in claim 72, wherein themetallic radioisotope is a radionuclide selected from the groupincluding ¹⁸⁶Re, ¹⁸⁸Re, ^(99m)Tc, ¹⁰⁵Rh, ¹⁹⁹Au, ¹⁵³Sm, ¹⁶⁶Ho and ⁹⁰Y.74. A method as set forth in claim 73, wherein said metallic isotopeincludes oxides and nitrides thereof.
 75. A method as set forth in claim57, wherein said chelating agent includes a multidentate chelatingstructure capable of forming a highly stable complex with metals viacoordinating atoms.
 76. A method as set forth in claim 75, wherein saidchelating structure includes the coordinating atoms S, N, O, or P.
 77. Amethod as set forth in claim 75, wherein the chelating structure is amacrocyclic compound including coordinating atoms.
 78. A method as setforth in claim 75, wherein said compound is a S₄ chelator.
 79. A methodas set forth in claim 75, wherein said compound is a N₄ chelator.
 80. Amethod as set forth in claim 75, wherein said compound is a N₂S₂chelator.
 81. A method as set forth in claim 75, wherein said compoundis a NS₃ chelator.
 82. A method as set forth in claim 75, wherein saidcompound is a N₃S chelator.
 83. A method as set forth in claim 57,wherein the compound has a binding affinity for the gastrin releasingpeptide receptor that is approximately equal to or greater than that ofnative bombesin.
 84. A method of imaging a tumor site by administeringto a subject a diagnostically effective amount of a compound as setforth in claim
 1. 85. A method of formulation a pharmaceutical using akit type method wherein the metal is added to a sealed vial containing apredetermined quantity of a compound described in claim 1 and a reducingagent.